Trans-activation Responsive Region Research Articles

Transfer of Tat and release of TAR RNA during the activation of the human immunodeficiency virus type-1 transcription elongation complex

The HIV-1 trans-activator protein, Tat, is a potent activator of transcriptional elongation. Tat is recruited to the elongating RNA polymerase during its transit through the trans-activation response region (TAR) because of its ability to bind directly to TAR RNA expressed on the nascent RNA chain. We have shown that transcription complexes that have acquired Tat produce 3-fold more full-length transcripts than complexes not exposed to Tat. Western blotting experiments demonstrated that Tat is tightly associated with the paused polymerases. To determine whether TAR RNA also becomes attached to the transcription complex, DNA oligonucleotides were annealed to the nascent chains on the arrested complexes and the RNA was cleaved by RNase H. After cleavage, the 5' end of the nascent chain, carrying TAR RNA, is quantitatively removed, but the 3' end of the transcript remains associated with the transcription complex. Even after the removal of TAR RNA, transcription complexes that have been activated by Tat show enhanced processivity. We conclude that Tat, together with cellular co-factors, becomes attached to the transcription complex and stimulates processivity, whereas TAR RNA does not play a direct role in the activation of elongation and is used simply to recruit Tat and cellular co-factors.

Evidence for a unique mechanism of strand transfer from the transactivation response region of HIV-1.

We previously found that strand transfer by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is promoted at sites where RT pauses during synthesis. In this report, strand transfer is measured within the 5' transactivation response region (TAR) of HIV-1 RNA. We hypothesized that the stable hairpin structure of TAR would induce RT pausing, promoting RNase H-directed cleavage of the template and subsequent transfer at that site. We further predicted that HIV-1 nucleocapsid protein (NC), known to melt secondary structures, would decrease transfer. We show that TAR created a strong pause site for RT, but NC significantly promoted strand transfer. The effect of NC is specific, since other single strand binding proteins failed to stimulate transfer. In another unexpected outcome, preferred positions of internal transfer were not at the pause site but were in the upper stem and loop of TAR. Thus, we propose a new mechanism for transfer within TAR described by an interactive hairpin model, in which association between the donor and the acceptor templates within the TAR stem promotes transfer. The model is consistent with the observed stimulation of strand transfer by NC. The model is applicable to internal and replicative end transfer.

Stable human immunodeficiency virus type 1 (HIV-1) resistance in transformed CD4+ monocytic cells treated with multitargeting HIV-1 antisense sequences incorporated into U1 snRNA.

We have approached the development of a human immunodeficiency virus type 1 (HIV-1) therapeutic product by producing immune cells stably resistant to HIV-1. Promonocytic CD4+ cells (U937) were made resistant to HIV-1 by the introduction of a DNA construct (pNDU1A,B,C) that contained three independent antisense sequences directed against two functional regions, transactivation response and tat/rev, of the HIV-1 target. Each sequence was incorporated into the transcribed region of a U1 snRNA gene to generate U1/HIV antisense RNA. Stably transfected cells expressed all three U1/HIV antisense transcripts, and these transcripts accumulated in the nucleus. These cells were subjected to two successive challenges with HIV-1 (BAL strain). The surviving cells showed normal growth characteristics and have retained their CD4+ phenotype. In situ hybridization assays showed that essentially all of the surviving cells produced U1/HIV antisense RNA. No detectable p24 antigen was observed, no syncytium formation was observed, and PCR-amplified HIV gag sequences were not detected. Rechallenge with HIV-1 (IIIB strain) similarly yielded no infection at a relatively high multiplicity of infection. As a further demonstration that the antisense RNA directed against HIV-1 was functioning in these transfected immune cells, Tat-activated expression of chloramphenicol acetyltransferase was shown to be specifically inhibited in cells expressing Tat and transactivation response region antisense sequences.

An NF-κB Site in the 5′-Untranslated Leader Region of the Human Immunodeficiency Virus Type 1 Enhances the Viral Expression in Response to NF-κB-activating Stimuli

The 5'-untranslated leader region of human immunodeficiency virus, type 1 (HIV-1), includes a complex array of putative regulatory elements whose role in the viral expression is not completely understood. Here we demonstrate the presence of an NF-kappaB-responsive element in the trans-activation response (TAR) region of HIV-1 that confers the full induction of HIV-1 long terminal repeat (LTR) in response to NF-kappaB-activating stimuli, such as DNA alkylating agents, phorbol 12-myristate 13-acetate, and tumor necrosis factor-alpha. The TAR NF-kappaB site GGGAGCTCTC spans from positions +31 to +40 and cooperates with the NF-kappaB enhancer upstream of the TATA box in the NF-kappaB-mediated induction of HIV-1 LTR. The conclusion stems from the following observations: (i) deletion of the two NF-kappaB sites upstream of the TATA box reduces, but does not abolish, the HIV-1 LTR activation by NF-kappaB inducers; (ii) deletion or base pair substitutions of the TAR NF-kappaB site significantly reduce the HIV-1 LTR activation by NF-kappaB inducers; (iii) deletions of both the NF-kappaB sites upstream of the TATA box and the TAR NF-kappaB site abolish the activation of HIV-1 LTR in response to NF-kappaB inducers. Moreover, the p50 p65 NF-kappaB complex binds to the TAR NF-kappaB sequence and trans-activates the TAR NF-kappaB-directed expression. The identification of an additional NF-kappaB site in the HIV-1 LTR points to the relevance of NF-kappaB factors in the HIV-1 life cycle.

Visualizing a Specific Contact in the HIV-1 Tat Protein Fragment and trans-Activation Responsive Region RNA Complex by Photocross-linking

Replication of human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the trans-activation responsive region (TAR) RNA, a stem-loop structure containing two helical stem regions separated by a trinucleotide bulge. The Tat protein contains a basic RNA-binding region (amino acids 49-57) located in the carboxyl-terminal half of the protein, and peptides containing this basic domain of Tat protein can bind TAR RNA with high affinities. We synthesized a 31-amino acid Tat fragment (amino acids 42-72) containing the basic region and part of flanking regulatory core domain that formed a specific complex with TAR RNA. Upon UV irradiation (254 nm), this Tat fragment cross-linked covalently with TAR RNA. Sites of cross-links were determined on both the TAR RNA and Tat protein fragment by RNA and protein sequencing, respectively. These results revealed that guanosine 26 of TAR RNA was cross-linked with tyrosine 47 of the Tat peptide. Our results provide the first physical evidence for a direct amino acid-base contact in Tat-TAR complex. Recently, orientation of the Tat-(42-72) was determined in our laboratory by psoralen.Tat-(42-72) conjugate (Wang, Z., and Rana, T. M. (1995) J. Am. Chem. Soc. 117, 5438-5444). On the basis of our findings, we suggest a model in which Tat binds to TAR RNA by inserting the basic recognition sequence into the major groove with an orientation where lysine 41 in the core domain of Tat contacts the lower stem and Tyr47 is close to G26 of TAR RNA. The knowledge of the orientation of Tat and details of other interactions with TAR RNA in Tat-TAR complex has significant implications for understanding gene regulation in HIV-1.

Efficient transactivation of the minute virus of mice P38 promoter requires upstream binding of NS1.

The P38 promoter of the autonomous parvovirus minute virus of mice is strongly transactivated by the nonstructural protein NS1, a sequence-specific DNA-binding protein. In the context of the complete viral genome, the only unique cis-acting signals required for P38 transactivation by NS1 are the proximal Sp1 site and the TATA element. In the absence of additional upstream sequences, a dependence upon the NS1 binding site within the transactivation response region is observed. Addition of synthetic NS1 binding sites to transactivation response region deletion mutants can restore the ability of NS1 to transactivate P38, and NS1 transactivation has been directly correlated to its ability to bind upstream of the P38 promoter.

RNA conformation in the Tat-TAR complex determined by site-specific photo-cross-linking.

Transcriptional regulation in human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the transactivation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5'-end of all mRNAs. We have used a site-specific cross-linking method based on 4-thiouracil (4-thioU) photochemistry to determine the conformation of TAR RNA and its interaction with Tat protein under physiological conditions. Three different TAR RNA constructs with a single 4-thioU residue at position 23, 38, or 40 were synthesized. Upon UV irradiation, 4-thioU at all three positions formed interstrand covalent cross-links in TAR RNA. Determination of cross-link sites by RNA sequencing revealed that 4-thioU at position 23 makes a direct contact with U40, while a 4-thoU at position 40 cross-links to C24 and C25, and at position 38, 4-thioU contacts G26 in TAR RNA. The addition of arginine did not alter the yield or the site of RNA-RNA cross-link. However, in the presence of Tat(38-72), UV irradiation of RNA modified with 4-thioU at position 23 or 38 resulted in RNA- protein cross-links, but no RNA-RNA cross-links were observed. 4-thioU at position 40 formed both RNA-RNA and RNA-protein cross-links in the presence of Tat(38-72). An intriguing finding of our studies was that a cross-linked TAR RNA with 4-thioU at position 40 retained specific Tat-binding activity. Our results establish four important conclusions about Tat-TAR structure. (1) U23 of free TAR RNA is in close contact with U40. (2) U40 is in close proximity to C24 and C25 both in free TAR RNA and in a complex with Tat. (3) Tat protein directly contacts U23, U38, and U40 in the major groove of TAR RNA. (4) Tat protein can recognize a TAR RNA structure containing an interrupted bulge which is formed by a covalent link between U40 and two bulge residues, C24 and C25. These structural studies provide new insights into tertiary folding of TAR RNA and its interaction with Tat protein.

High-resolution mapping of the human T-cell leukemia virus type 1 Rex-binding element by in vitro selection.

Interactions between the Rex protein of HTLV-1 and the genomic Rex-binding element (XBE) mediate the cytoplasmic transport of viral mRNAs. However, it is uncertain which RNA sequences and structures contribute to Rex recognition. A portion of the viral genome that spanned the XBE was partially randomized, and functional Rex-binding variants were selected. Alignment of selected Rex-binding sequences revealed positions that were functionally conserved between different molecules. A model is presented in which a subset of the selected residues are in direct contact with Rex. Positions that covaried with one another were also found. These covariations support a secondary-structural model in which a central paired stem is symmetrically flanked by two bulge loops. On the basis of this model, site-directed mutations of the XBE were constructed and each half molecule was found to bind independently to Rex. The functional residues and secondary structures in the XBE half molecules bear a remarkable resemblance to the transactivation response region element of HIV-1. Since the transactivation response region element is known to interact specifically with arginine residues in the Tat protein, these results suggest that the XBE binds to the arginine-rich RNA-binding domain of Rex in a similar manner. This model is supported by the selection data.

RNA-binding Proteins That Specifically Recognize the Selenocysteine Insertion Sequence of Human Cellular Glutathione Peroxidase mRNA

Translational incorporation of the unusual amino acid selenocysteine in eukaryotes requires a coding region UGA codon (which otherwise serves as a termination signal), a selenocysteine insertion sequence (SECIS) in the 3'-untranslated region of the mRNA, and selenocysteyl-tRNA. The mechanisms involved in SECIS recognition by the eukaryotic translational machinery remain unknown. We report the detection of RNA-binding proteins that specifically recognize the SECIS from human cellular glutathione peroxidase (GPX1) transcripts. RNA gel shift assays showed three retarded bands after incubation with COS-1 whole cell lysate or S-100 cytosol fraction or with extracts from hepatoma cell lines HepG2 and Hep3B. The specificity of the binding was demonstrated by competition by cold unlabeled SECIS RNA and by lack of competition by other RNA species with similar stem-loop secondary structures, such as the human immunodeficiency virus (HIV) transactivation-response region of HIV mRNA element, and mutated SECIS constructs. UV cross-linking and SDS-polyacrylamide gel electrophoresis revealed at least two proteins, with estimated molecular masses of 55,000 and 65,000 Da, that bind to the SECIS. Examination of a series of insertion and deletion SECIS mutants indicated recognition of the SECIS primarily through the basal stem region, although the upper stem, loop, and two of three short conserved sequences also appear to contribute to the affinity of the binding.

The Structure of the Human Immunodeficiency Virus Type-1 TAR RNA Reveals Principles of RNA Recognition by Tat Protein

The human immunodeficiency virus type-1 (HIV-1) Tat protein stimulates transcriptional elongation. Tat is introduced to the transcription machinery by binding to the transactivation response region (TAR) RNA stem-loop encoded by the 5′ leader sequence found on all HIV-1 mRNAs. We have used multidimensional heteronuclear NMR to determine the structure of the TAR RNA in the presence of the ADP-1 polypeptide, a 37-mer that carries the minimal RNA recognition region of the Tat protein and closely mimics Tat binding specificity, In the presence of a variety of ligands, including ADP-1, related basic peptides and the amino acid derivative argininamide, the bulge region of TAR undergoes a local conformational rearrangement and forms a more stable structure. The structure of TAR in the bound form has been determined from over 1000 NMR-derived constraints. The U23 residue at the 5′ end of the bulge is positioned near G26 and A27 in the major groove, rather than stacked on A22 as in the free TAR. U23 and G26 are brought into close proximity by contacts to the guanidinium group and side-chain amide group of a common arginine residue. However, the interaction of this guanidinium group with TAR is not the only source of binding specificity. Besides NOEs to the arginine residue participating in the conformational change, ADP-1 shows additional intermolecular NOEs to TAR suggesting that there are multiple points of contacts between TAR RNA and residues from the basic and core regions of Tat. These structural results provide important clues towards the identification of small molecular mass and/or peptidomimetic inhibitors of the essential Tat-TAR interaction.

The trans-inhibitory Rep78 protein of adeno-associated virus binds to TAR region DNA of the human immunodeficiency virus type 1 long terminal repeat

]he large rep gene products, Rep78 and Rep68, of adeno-associated virus (AAV) are pleiotropic effector proteins which are required for AAV DNA replication and the trans-regulation of AAV gene expression. Apart from these essential functions prerequisite for the life cycle of AAV, these rep products are able to inhibit the replication and gene expression of human immunodeficiency virus type 1 (HIV-1) and a number of DNA viruses. Here, it is demonstrated that Rep78, as a chimeric with the maltose binding protein, directly binds the full-length HIV-1 long terminal repeat (LTR), and to a subset of these sequences containing the trans-activation response (TAR) sequence as DNA. These interactions, an effector protein physically binding a target promoter, suggest a direct mechanism of action for Rep78 inhibition. Furthermore, competitive binding studies between the TAR region and the full-length HIV-LTR, strongly suggested that another site(s) within the LTR was also bound by Rep78. Finally, as Rep78 binding is also believed to be affected by secondary structure within the DNA, it was found that Rep78 preferentially binds with HIV-LTR sequences with promoted secondary structure generated by heat denaturation and rapid cooling.

Nucleotide sequence analysis of Jembrana disease virus: a bovine lentivirus associated with an acute disease syndrome.

The complete nucleotide sequence of the RNA genome of Jembrana disease virus (JDV), a lentivirus that causes an acute disease syndrome in Bali cattle (Bos javanicus), is reported. In addition to the gag, pol and env genes and flanking long terminal repeats (LTRs) that characterize all retroviruses, a number of accessory genes represented by small multiply spliced ORFs in the central and 3'-terminal regions of the genome, including tat and rev that are typical of lentiviruses, were identified. The genome of JDV was 7732 bp in length, 750 bp smaller than the genome of bovine immunodeficiency virus (BIV) strain BIV127. A striking feature of the genome was the many deletions relative to BIV127, the largest of which were 471 bp from the env gene and 157 bp from the U3 (promoter) region in the LTR. There were also several insertions of up to 33 bp in the JDV genome relative to BIV127 found in the env gene and small ORFs that overlap env. Other significant genomic differences between JDV and BIV127 included changes to cis-acting sequences throughout the genome such as promoter and enhancer sequences in the LTR, the trans-activation response region, splice sites and frameshift sequences; alterations to the gag precursor protein cleavage sites and thus the processed products; loss of the vpw and vpy ORFs; and amino acid changes in all coding regions. The significance of these changes is discussed in relation to the differences in pathogenicity between JDV and BIV.

Bulge-Specific Cleavage in Transactivation Response Region RNA and Its DNA Analog by Neocarzinostatin Chromophore

On the basis of the finding that in the absence of thiol the nonprotein chromophore of the antitumor drug neocarzinostatin (NCS-chrom) induces highly efficient site-specific cleavage at a single site on the 3' side of a bulge in single-stranded DNA involving entirely 5' chemistry [Kappen, L. S., & Goldberg, I. H. (1993) Science, 261, 1319-1321], transactivation response region (TAR) RNA (29-mer) and its DNA analogue which presumably contain bulge structures were tested as potential substrates for NCS-chrom. In TAR RNA NCS-chrom generates a distinct but weak band due to cleavage at U24 in the bulge. Cleavage at U24 has a pH dependence and time course similar to those for previously studied DNA bulges. This band is not produced in drug reactions containing glutathione, by the protein component of native NCS, or by inactivated NCS-chrom. Cleavage at U24, albeit weak, occurs in an RNA substrate made up of two linear RNA oligomers which presumably can form a bulge akin to that in TAR RNA. In the DNA analogue of TAR RNA, as well as in a DNA duplex made of two linear oligomers that can form a similar bulge, NCS-chrom causes strand cleavage at the T residues in the bulge and at the bases flanking the bulge. Cleavage at T25 in the bulge involves, in addition to 5' chemistry, 4' attack which results in a fragment with mobility characteristic of 3'-phosphoglycolate-ended fragments. Experiments using DNA substrate having deuterium selectively at the 4' or 5' positions of T25 confirm 4' attack and show kinetic shuttling between the two positions.(ABSTRACT TRUNCATED AT 250 WORDS)

Major groove opening at the HIV-1 Tat binding site of TAR RNA evidenced by a rhodium probe.

Transactivation of human immunodeficiency virus (HIV) gene expression requires the interaction of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5'-end of all mRNAs. The TAR RNA contains a six-nucleotide loop and a three-nucleotide pyrimidine bulge which separates two helical stem regions. The trinucleotide bulge is essential for high affinity and specific binding of the Tat protein. Recently, a rhodium complex, Rh(phen)2phi3+, was discovered which promotes RNA cleavage in the open major groove and triply bonded bases [Chow, C. S., et al. (1992) Biochemistry 31, 972-982]. This metal complex does not bind double-helical RNA or unstructured single-stranded regions of RNA. Instead, sites of tertiary interaction which are open in the major groove and accessible to stacking are targeted by the complex through photoactivated cleavage. We have used this rhodium probe to investigate the effect of bulge bases on the major groove opening in TAR RNA. The sites targeted by the rhodium complex have been mapped to single nucleotide resolution on wild-type TAR RNA and on several mutants of the TAR RNA containing different numbers of mismatch bases in the bulge region. A strong cleavage at residues C39 and U40 was observed on the wild-type TAR RNA and in mutant TAR RNA containing two mismatch bases in the bulge. No cleavage at C39 and U40 was observed in a bulgeless and a one-base bulge TAR RNA. By varying the number of mismatch bases, we demonstrated that the trinuclear bulge widens the major groove of TAR RNA to facilitate Tat binding.(ABSTRACT TRUNCATED AT 250 WORDS)

The RNA element encoded by the trans-activation-responsive region of human immunodeficiency virus type 1 is functional when displaced downstream of the start of transcription.

The human immunodeficiency virus type 1 (HIV-1) trans-activator protein, Tat, specifically stimulates transcription from the viral long terminal repeat. Tat binds to an RNA stem-loop structure encoded by the trans-activation response region (TAR). To test whether TAR is functional when displaced downstream of the start of transcription, we assayed a series of templates carrying duplicated TAR elements in cell-free transcription systems. When the normally positioned TAR element (TAR-1) is inactivated by mutations in either the Tat binding site or the apical loop sequence, which acts as the binding site for a cellular factor, transactivation can be rescued by a wild-type TAR element placed downstream (TAR-2). The TAR-2 element is functional even when placed > 200 nt downstream of TAR-1. TAR complementation experiments have also shown that a functional TAR element requires both an intact Tat binding site and an intact apical loop sequence. For example, if TAR-1 carries a mutation in the loop element it cannot be rescued by a TAR-2 element carrying a mutation in the Tat binding site. Substitution mutations in TAR-1 show that the 5' half of TAR also encodes an essential DNA element which is required for efficient transcription initiation. These results strongly suggest that Tat and cellular cofactors which bind TAR RNA associate with the transcription complex during its transit through TAR.

Methylphosphonate mapping of phosphate contacts critical for RNA recognition by the human immunodeficiency virus tat and rev proteins.

The HIV-1 regulatory proteins tat and rev are both RNA binding proteins which recognize sequences in duplex RNA which are close to structural distortions. Here we identify phosphate contacts which are critical for each binding reaction by use of a new method. Model RNA binding sites are constructed carrying substitutions of individual phosphodiesters by uncharged methylphosphonate derivatives isolated separately as Rp and Sp diastereoisomers and tested for protein binding by competition assays. In the binding of tat to the trans-activation response region (TAR), three phosphates, P21 and P22 which are adjacent to the U-rich bulge and P40 on the opposite strand, are essential and in each case both isomers inhibit binding. Similarly, in the interaction between the HIV-1 rev protein and the rev-responsive element (RRE) both methylphosphonate isomers at P103, P104, P124 and P125 interfere with rev binding. At P106, only the Rp methylphosphonate isomer is impaired in rev binding ability and it is proposed that the Rp oxygen is hydrogen-bonded to an uncharged amino acid or to a main chain hydrogen atom. Synthetic chemistry techniques also provide evidence for the conformations of non-Watson-Crick G106:G129 and G105:A131 base-pairs in the RRE 'bubble' structure upon rev binding. Almost all functional groups on the 5 bulged residues in the bubble have been ruled out as sites of contact with rev but, by contrast, the N7-positions of each G residue in the flanking base-pairs are identified as sites of likely hydrogen-bonding to rev. The results show that both tat and rev recognize the major groove of distorted RNA helixes and that both proteins make specific contacts with phosphates which are displaced from the sites of base-pair contact.

Human immunodeficiency virus type 1 transactivator protein, tat, stimulates transcriptional read-through of distal terminator sequences in vitro.

The human immunodeficiency virus type 1 transactivator protein, tat, specifically stimulates transcription from the viral long terminal repeat. We used cell-free transcription systems to test whether tat can stimulate transcriptional read-through of an artificial terminator sequence (e.g., a stable RNA stem-loop structure followed by a tract of nine uridine residues) placed downstream of the viral long terminal repeat. In the absence of tat, RNA polymerases are prematurely released from the template at the terminator sequence. Recombinant tat protein purified from Escherichia coli increased the synthesis of full-length transcripts approximately 25-fold and decreased the amount of transcripts ending at the terminator sequence. The reaction is strictly dependent upon the presence of a functional transactivation-responsive region (TAR) sequence. Mutations in the tat binding site on TAR RNA and mutations in the TAR RNA loop block transactivation in vivo. Neither type of mutation is able to respond to tat in vitro. These results strongly suggest that after transcription through the TAR region, tat modifies the transcription complex to increase its elongation capacity.

Two strains of SIV mac show differential transactivation mediated by sequences in the promoter

Two infectious molecular clones of simian immunodeficiency virus, SIV mac251 and SIV mac239, have very different in vivo properties, SIV mac239 being much more pathogenic than SIV mac251. To assess whether the in vivo differences between the two viruses would be reflected in transcriptional rates in vitro, transcriptional activity in the presence of the transactivation protein tat was analyzed by transient transfection assays in HUT-78 and U937 cells. Whereas the two promoters had similar basal activities ( Anderson and Clements, 1991, J. Virol. 65, 51–60) the promoter of SIV mac239 was transactivated to a greater extent. Removal of sequences 5′ to −225 and 3′ to +18 maintained the basal activity, yet made the promoter unresponsive to tat. Addition of bases +19 to +149 reconstituted transactivation and decreased basal activity. Analysis of deletion mutants with reconstituted transactivation response region determined that differences between the two strains were maintained even when only the proximal sequences, −225 to +18 of the U3 and R region were placed upstream of the TAR sequences. This region contains four nucleotide differences and the potential Sp-1-binding sites, where there are an additional 11 bases in SIV mac239 that create a third potential Sp-1 site, compared to only 2 in SIV mac251. Transactivation in this assay system was found to correlate better to RNA differences shortly after transfection (12 hr) than later (46 hr).

Human immunodeficiency virus type 1 Tat-mediated trans activation correlates with the phosphorylation state of a cellular TAR RNA stem-binding factor.

Protein kinase C (PKC) is involved in the mitogenic stimulation of cell proliferation and has recently been reported to be essential for Tat-mediated trans activation. We have determined that RNA binding of a cellular factor which specifically interacts with the trans-activation response region (TAR) is blocked in cells depleted of PKC activity by chronic phorbol myristate acetate stimulation. We also show that nuclear extracts can be depleted of the cellular TAR-binding factor by in vitro treatment with purified protein phosphatase 2A. Furthermore, TAR RNA-binding activity can be partially restored to depleted nuclear extracts in vitro by addition of PKC. Chimeric constructs in which the Tat protein is artificially tethered to viral RNA show PKC independence for Tat-mediated trans activation. Specific mutations in the TAR RNA stem region which cause reduced binding of host cell factor in vitro also cause reduced Tat-mediated trans activation in vivo. Together, these results suggest that phosphorylation-dependent binding of a cellular cofactor to TAR RNA is an essential step in Tat-mediated trans activation. Deciphering the regulation of Tat-mediated trans activation by phosphorylation will be critical in fully understanding the regulation of human immunodeficiency virus type 1 activation.

Synergistic activation of the human immunodeficiency virus type 1 promoter by the viral Tat protein and cellular transcription factor Sp1.

We have previously shown that the human immunodeficiency virus type 1 (HIV-1) Tat protein can activate a synthetic promoter containing consensus-binding sites for the cellular transcription factor Sp1. In this report, we show that a GAL-Tat fusion protein targeted via GAL4 DNA-binding sites can also trans activate an HIV-1 LTR promoter independently of the trans-activation response region. To show that the trans activation of the promoter by Tat directly involves the Sp1 protein, we have targeted a GAL-Sp1 fusion protein to the long terminal repeat promoter via upstream GAL4-binding sites. In the presence of Tat and GAL-Sp1, the promoter is synergistically trans activated at the transcriptional level, indicating that Tat and Sp1 functionally interact to trans activate the HIV-1 promoter. The Sp1 synergism is relatively specific, since another chimeric transcriptional activator, GAL-VP16, does not appear to be significantly synergistic with Tat.